Trends of insecticide resistance monitoring in mainland Tanzania, 2004-2020.

BACKGROUND: Insecticide resistance is a serious threat to the continued effectiveness of insecticide-based malaria vector control measures, such as long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS). This paper describes trends and dynamics of insecticide resistance and its underlying mechanisms from annual resistance monitoring surveys on Anopheles gambiae sensu lato (s.l.) populations conducted across mainland Tanzania from 2004 to 2020. METHODS: The World Health Organization (WHO) standard protocols were used to assess susceptibility of the wild female An. gambiae s.l. mosquitoes to insecticides, with mosquitoes exposed to diagnostic concentrations of permethrin, deltamethrin, lambdacyhalothrin, bendiocarb, and pirimiphos-methyl. WHO test papers at 5× and 10× the diagnostic concentrations were used to assess the intensity of resistance to pyrethroids; synergist tests using piperonyl butoxide (PBO) were carried out in sites where mosquitoes were found to be resistant to pyrethroids. To estimate insecticide resistance trends from 2004 to 2020, percentage mortalities from each site and time point were aggregated and regression analysis of mortality versus the Julian dates of bioassays was performed. RESULTS: Percentage of sites with pyrethroid resistance increased from 0% in 2004 to more than 80% in the 2020, suggesting resistance has been spreading geographically. Results indicate a strong negative association (p = 0.0001) between pyrethroids susceptibility status and survey year. The regression model shows that by 2020 over 40% of An. gambiae mosquitoes survived exposure to pyrethroids at their respective diagnostic doses. A decreasing trend of An. gambiae susceptibility to bendiocarb was observed over time, but this was not statistically significant (p = 0.8413). Anopheles gambiae exhibited high level of susceptibility to the pirimiphos-methyl in sampled sites. CONCLUSIONS: Anopheles gambiae Tanzania's major malaria vector, is now resistant to pyrethroids across the country with resistance increasing in prevalence and intensity and has been spreading geographically. This calls for urgent action for efficient malaria vector control tools to sustain the gains obtained in malaria control. Strengthening insecticide resistance monitoring is important for its management through evidence generation for effective malaria vector control decision.

Leishmania detection in sand flies using a field-deployable real-time analytic system.

We describe here the development and evaluation of advanced vector surveillance analytic technologies for real-time leishmaniasis risk assessment. Leishmania genus and visceral leishmaniasis causative agent--specific dual fluorogenic-probe hydrolysis (TaqMan), thermally stable (freeze-dried) polymerase chain reaction assays were developed using field-durable analytic instrumentation. In laboratory testing with a panel of diverse Leishmania species from culture and infected sand flies, the sensitivity and specificity of both assays were 100% concordant with DNA sequencing. In specificity testing with Leishmania genetic near neighbors, clinically significant organisms, and human genomic DNA, no detectable fluorescence above background was observed. Field evaluation was conducted in southern Iraq using wild sand flies. In field testing, Leishmania genus assay was 100% sensitive and 96% specific with a single false-positive result. The visceral leishmaniasis genotype assay was 100% sensitive and 100% specific compared to DNA sequencing. Thermally stable polymerase chain reaction assays vastly simplified transportation and storage. Assay preparation and analysis required less than 2 hours.

Use of vector diagnostics during military deployments: recent experience in Iraq and Afghanistan.

Vector-borne diseases such as malaria, dengue, and leishmaniasis are a threat to military forces deployed outside of the United States. The availability of specific information on the vector-borne disease threat (e.g., presence or absence of a specific disease agent, temporal and geographic distribution of competent vectors, and vector infection rates) allows for effective implementation of appropriate measures to protect our deployed military forces. Vector diagnostics can provide critical, real-time information crucial to establishing effective vector prevention/control programs. In this article we provide an overview of current vector diagnostic capabilities, evaluate the use of vector diagnostics in Operation Enduring Freedom and Operation Iraqi Freedom, and discuss the concept of operations under which vector diagnostics are employed.

Betalain rich functional extract with reduced salts and nitrate content from red beetroot (Beta vulgaris L.) using membrane separation technology.

An initial laboratory-scale evaluation of separation characteristics of membranes with nominal molecular weight cut-offs (NMWCO) ranging from 30kD down to 0.5kD indicated effective separation of betalains in the 0.5kD region. Subsequent pilot-level trials using 1kD, loose reverse osmosis (LRO) and reverse osmosis (RO) spiral-wound membranes showed LRO membrane to be very efficient with up to 96% salt and 47% other dissolved solids removed while retaining majority of the pigment (∼98%) in the betalain rich extract (BRE). The total betalain content in the BRE increased up to 46%, the highest recovery reported so far at pilot scale level. Interestingly, more than 95% of the nitrates were removed from the BRE after the three diafiltrations. These studies indicate that membrane technology is the most efficient technique to produce BRE with highly reduced amounts of salts and nitrate content.

Indoor residual spraying with micro-encapsulated pirimiphos-methyl (Actellic® 300CS) against malaria vectors in the Lake Victoria basin, Tanzania.

BACKGROUND: The indoor residual spraying programme for malaria vectors control was implemented in four districts of the Lake Victoria basin of Tanzania namely Ukerewe, Sengerema, Rorya andSerengeti. Entomological monitoring activities were implemented in one sentinel village in each district to evaluate the efficacy of pirimiphos-methyl 300 CS sprayed on different wall surfaces and its impact against malaria vectors post-IRS intervention. METHODS: The residual decay rate of p-methyl 300 CS applied at a target dosage of 1g a.i./m2 on thesprayed wall surfaces was monitored for a period of 43 weeks post-IRSusing the WHO cone wall bioassay method. The bioassays were performed by exposing 2-5 days old unfed susceptible female Anopheles gambiae s.s. (Kisumu strain) to sprayed wall surfaces for a period of 30 minutes. In each sentinel village, mosquito collection was carried out by trained community mosquito collectors. Monthly mosquito collections were carried out from 6.00pm to 6.00am using CDC light traps and clay pot methods for indoors host seekingand outdoors resting mosquitoes respectively. Six traps (2 CDC light traps and 4 clay pots) were set per sentinel village per night for28 consecutive days in a moon. PCR and ELISA were used for mosquito species identification and sporozoite detection, respectively. RESULTS: Based on the WHOPES recommendation, insecticides should have a minimum efficacy of ≥ 80% mosquito mortality at 24 hours post exposure on the sprayed wall surfaces to be considered effective. In this study, p-methyl 300 CS was demonstrated to have a long residual efficacy of 21-43 weeks post-IRS on mud, cement, painted and wood wall surfaces. Numberof anopheline mosquitoes decreased post-IRS interventions in all sentinel villages. The highest numbers ofanopheline mosquitoes were collected in November-December, 38-43 weeks post-IRS. A total of 270 female anopheline mosquitoes were analyzed by PCR; out of which 236 (87.4%) were An. gambiae s.l. and 34 (12.6%) were An. funestus group. Of the 236 An. gambiae s.l.identified 12.6% (n = 34) were An. gambiae s.s. and 68.6% (n = 162) were An. arabiensis. Ofthe 34 An. funestus group indentified 91.2% (n = 31) were An. parensis and 8.8% (n = 3) were An. rivulorum. The overall Plasmodium falciparum sporozoite rate was 0.7% (n = 2,098). CONCLUSIONS: Pirimiphos-methyl 300 CS was found to be effective for IRS in the Lake Victoria basin,Tanzania. P-methyl 300 CShas a long residual efficacy on sprayed wall surfaces and therefore it is effective in controlling principal malaria vectors of An. gambiae s.l and An. funestus which rest on wall surfaces after and before feeding.

Epidemiology and spatial analysis of malaria in the Northern Peruvian Amazon.

A retrospective surveillance study was conducted to examine the micro-geographic variation of malaria incidence in three malaria-endemic communities in the Northern Peruvian Amazon. The annual malaria risk rate (per 100) ranged from 38% to 47% for Plasmodium vivax and from 15% to 18% for P. falciparum. Spatial clusters were found for P. vivax in Padre Cocha, Manacamiri, and Zungaro Cocha, and for P. falciparum only in Padre Cocha. Spatial-temporal clusters showed that the highest monthly number of P. vivax cases varied every year from December to March in 1996-1997 and from February to June in 1998-1999, and for P. falciparum from November to April in 1996-1997 and from January to April in 1998-1999. Our results suggest a constant presence of high-risk areas (hot spots) for malaria infection in periods with high or low malaria incidence. Modest targeted control efforts directed at identified high-risk areas may have significant impact on malaria transmission in this region.

Spatial clustering and epidemiological aspects of visceral leishmaniasis in two endemic villages, Baringo District, Kenya.

Visceral leishmaniasis (VL) seroprevalence in Kenya is unknown because of the lack of a practical and accurate diagnostic test or surveillance system. A novel serological assay was used to estimate the seroprevalence of Leishmania-specific antibodies, and Global Information System and spatial clustering techniques were applied to study the presence of spatial clusters in Parkarin and Loboi villages in Baringo District in 2001. VL seroprevalences were 52.5% in Parkarin and 16.9% in Loboi. Significant associations among seropositivity and house construction, age, and proximity to domestic animal enclosures were found. A significant spatial cluster of VL was found in Loboi. The spatial distribution of cases in the two villages was different with respect to risk factors, such as presence of domestic animals. This study suggests that disease control efforts could be focused on elimination of sand fly habitat, placement of domestic animal enclosures, and targeted use of insecticides.

Circulating brain derived neurotrophic factor (BDNF) and frequency of BDNF positive T cells in peripheral blood in human ischemic stroke: Effect on outcome.

The aim of this study was to measure the levels of circulating BDNF and the frequency of BDNF-producing T cells after acute ischaemic stroke. Serum BDNF levels were measured by ELISA. Flow cytometry was used to enumerate peripheral blood leukocytes that were labelled with antibodies against markers of T cells, T regulatory cells (Tregs), and intracellular BDNF. There was a slight increase in serum BDNF levels after stroke. There was no overall difference between stroke patients and controls in the frequency of CD4(+) and CD8(+) BDNF(+) cells, although a subgroup of stroke patients showed high frequencies of these cells. However, there was an increase in the percentage of BDNF(+) Treg cells in the CD4(+) population in stroke patients compared to controls. Patients with high percentages of CD4(+) BDNF(+) Treg cells had a better outcome at 6months than those with lower levels. These groups did not differ in age, gender or initial stroke severity. Enhancement of BDNF production after stroke could be a useful means of improving neuroprotection and recovery after stroke.

Bed Net Durability Assessments: Exploring a Composite Measure of Net Damage.

BACKGROUND: The durability of Long Lasting Insecticidal Nets (LLINs) in field conditions is of great importance for malaria prevention and control efforts; however, the physical integrity of the net fabric is not well understood making it challenging to determine overall effectiveness of nets as they age. The 2011 World Health Organization Pesticide Evaluation Scheme (WHOPES) guidelines provide a simple, standardized method using a proportional hole index (PHI) for assessing net damage with the intent to provide national malaria control programs with guidelines to assess the useful life of LLINS and estimate the rate of replacement. METHODS: We evaluated the utility of the PHI measure using 409 LLINs collected over three years in Nampula Province, Mozambique following a mass distribution campaign in 2008. For each LLIN the diameter and distance from the bottom of the net were recorded for every hole. Holes were classified into four size categories and a PHI was calculated following WHOPES guidelines. We investigate how the size, shape, and location of holes influence the PHI. The areas of the WHOPES defined categories were compared to circular and elliptical areas based on approximate shape and actual measured axes of each hole and the PHI was compared to cumulative damaged surface area of the LLIN. RESULTS: The damaged area of small, medium, large, and extra-large holes was overestimated using the WHOPES categories compared to elliptical areas using the actual measured axes. Similar results were found when comparing to circular areas except for extra-large holes which were underestimated. (Wilcoxon signed rank test of differences p< 0.0001 for all sizes). Approximating holes as circular overestimated hole surface area by 1.5 to 2 times or more. There was a significant difference in the mean number of holes < 0.5 cm by brand and there were more holes of all sizes on the bottom of nets than the top. For a range of hypothetical PHI thresholds used to designate a "failed LLIN", roughly 75 to 80% of failed LLINs were detected by considering large and extra-large holes alone, but sensitivity varied by brand. CONCLUSIONS: Future studies may refine the PHI to better approximate overall damaged surface area. Furthermore, research is needed to identify whether or not appropriate PHI thresholds can be used to deem a net no longer protective. Once a cutoff is selected, simpler methods of determining the effective lifespan of LLINs can help guide replacement strategies for malaria control programs.

Physical durability of two types of long-lasting insecticidal nets (LLINs) three years after a mass LLIN distribution campaign in Mozambique, 2008-2011.

We conducted a prospective evaluation to measure the physical durability of two brands of long-lasting insecticidal nets (LLINs) distributed during a campaign in 2008 in Nampula Province, Mozambique. Households with LLINs tagged during the campaign (6,000) were geo-located (34%) and a random sample was selected for each of 3 years of follow-up. The LLINs were evaluated in the field and a laboratory for presence of holes and a proportional hole index (pHI) was calculated following the World Health Organization guidelines. We performed 567 interviews (79.0%) and found 75.3% (72.1-78.4%) of households retained at least one LLIN after 3 years; the most common cause of attrition was damage beyond repair (51.0%). Hole damage was evident after 1 year, and increased by year. Olyset had a significantly greater mean number of holes and pHI compared with PermaNet 2.0 brand (all P values ≤ 0.001). Additional information about LLIN durability is recommended to improve malaria control efforts.

Evaluation of serological assays based on a novel excreted antigen preparation for the diagnosis of Cutaneous Leishmaniasis in Panama.

The objective of the present study was to determine the efficacy of prototype diagnostic serological assays for American Cutaneous Leishmaniasis (ACL) in Panama. As such, we prospectively sampled 100 cutaneous leishmaniasis case-patients and tested their sera in two serological assays based upon novel soluble antigen preparations made from propagating the parasites in a protein-free, serum free media. Using serum and a Leishmania mexicana antigen preparation to sensitize plates, the assay correctly identified 89% of the case-patients. While using serum with an antigen preparation from Leishmania braziliensis, the assay correctly identified 71% of the patients. Concerning both test formats, performance was near equal in true positive and presumptive positive subsets demonstrating the improved sensitivity of these assays over reference methods of choice. Since the incidence of leishmaniasis in Panama has increased dramatically in the past 10 years, these assays may be useful in clinical and epidemiological studies and control programs.

Evaluation of a dipstick malaria sporozoite panel assay for detection of naturally infected mosquitoes.

The determination of the presence or absence of malaria sporozoites in wild-caught Anopheles mosquitoes remains an integral component to the understanding of the transmission dynamics in endemic areas. To improve that capability, there has been on-going development of a new device using dipstick immunochromatographic technology for simplifying the testing procedure and reducing the time required to obtain results. As part of a larger multi-center effort, we evaluated the sensitivity and specificity of a prototype malaria sporozoite antigen panel assay (Medical Analysis Systems, Camarillo, CA) against three human Plasmodium species/polymorphs. The wicking (dipstick) assay was compared against a standard parasite antigen capture enzyme-linked immunosorbent assay (ELISA) for the detection of human circumsporozoite protein (CSP) in wild-caught mosquitoes. Over 6,800 Anopheles mosquitoes, representing 20 species collected from malaria endemic areas of Indonesia were tested either individually or in pools of up to 10 mosquitoes each. From 1,442 pooled test strip assays and ELISA formats, nine mosquito pools were found reactive for P.falciparum, P. vivax 210, or P. vivax 247 CSP. There was complete concordance between test strip results and ELISA results. Sensitivity was 100% and given some minor problems with false positives or negatives, specificity (n = 488) was 97%. Most strips judged as false positive produced very weak signals compared with negative control blank strips and paired ELISA-negative samples. The dipstick test proved technically simpler to perform and interpret than the ELISA and results were obtained within 15 min of exposure to mosquito suspension. This qualitative assay appears an attractive alternative to the CSP ELISA for detection of sporozoites in fresh or dried mosquitoes.

Evaluation of the VecTest Malaria Antigen Panel assay for the detection of Plasmodium falciparum and P. vivax circumsporozoite protein in anopheline mosquitoes in Thailand.

We evaluated the performance of the VecTest Malaria Antigen Panel (V-MAP) assay for the detection of Plasmodium falciparum and P. vivax (variants 210 and 247) circumsporozoite protein in anopheline mosquitoes in Thailand. The V-MAP assay is a rapid, one-step procedure using a 'dipstick' wicking test strip. The circumsporozoite (CS) ELISA was used as the reference standard. Mosquitoes evaluated in the study included field-collected specimens (n = 930) and laboratory-reared specimens that had been fed on blood collected from patients with and without Plasmodium gametocytes (n = 4,110) or on cultured P. falciparum gametocytes (n = 262). Field-collected mosquitoes were triturated individually or in pools of 2-5 and tested using 613 V-MAP assays. Laboratory-reared specimens were tested individually using 4,372 V-MAP assays. Assay performance depended on the species of Plasmodium and the number of sporozoites used as the cut-off. For P. falciparum, optimal performance was achieved using a cut-off of 150 sporozoites (sensitivity = 100%, specificity = 99.2%, and accuracy = 0.99). For P. vivax variant 210, optimal performance was also achieved using a cut-off of 150 sporozoites (sensitivity = 94.8%, specificity = 94.5%, and accuracy = 0.95). We were unable to develop a standard-curve for the CS-ELISA using P. vivax variant 247 because of a lack of sporozoites; however, using a cut-off of 30 pg P. vivax 247 antigen (mosquitoes with less than this amount of antigen were considered negative), assay performance (sensitivity = 94.3%, specificity = 99.2%, and accuracy = 0.99) was comparable to that achieved for P. falciparum and P. vivax 210. These results clearly demonstrate that the V-MAP assay performs at an acceptable level and offers practical advantages for field workers needing to make rapid surveys of malaria vectors.

Modular laboratories--cost-effective and sustainable infrastructure for resource-limited settings.

High-quality laboratory space to support basic science, clinical research projects, or health services is often severely lacking in the developing world. Moreover, the construction of suitable facilities using traditional methods is time-consuming, expensive, and challenging to implement. Three real world examples showing how shipping containers can be converted into modern laboratories are highlighted. These include use as an insectary, a molecular laboratory, and a BSL-3 containment laboratory. These modular conversions have a number of advantages over brick and mortar construction and provide a cost-effective and timely solution to offer high-quality, user-friendly laboratory space applicable within the developing world.

Immunization with Leishmania major exogenous antigens protects susceptible BALB/c mice against challenge infection with L. major.

The potential of Leishmania major culture-derived soluble exogenous antigens (SEAgs) to induce a protective response in susceptible BALB/c mice challenged with L. major promastigotes was investigated. Groups of BALB/c mice were immunized with L. major SEAgs alone, L. major SEAgs coadministered with either alum (aluminum hydroxide gel) or recombinant murine interleukin-12 (rmIL-12), L. major SEAgs coadministered with both alum and rmIL-12, and L. major SEAgs coadministered with Montanide ISA 720. Importantly and surprisingly, the greatest and most consistent protection against challenge with L. major was seen in mice immunized with L. major SEAgs alone, in the absence of any adjuvant. Mice immunized with L. major SEAgs had significantly smaller lesions that at times contained more than 100-fold fewer parasites. When lymphoid cells from L. major SEAg-immunized mice were stimulated with leishmanial antigen in vitro, they proliferated and secreted a mixed profile of type 1 and type 2 cytokines. Finally, analyses with Western blot analyses and antibodies against three surface-expressed and secreted molecules of L. major (lipophosphoglycan, gp46/M2/PSA-2, and gp63) revealed that two of these molecules are present in L. major SEAgs, lipophosphoglycan and the molecules that associate with it and gp46/M2/PSA-2.